Several methods are known from the state of the art for detection and identification of microorganisms. Optical methods are particularly popular amongst the various detection methods due to their high speed and the low preparation efforts they require.
One way to detect and identify microorganisms is based on the evaluation of inelastically scattered light from the specimen. This so-called Raman scattering can be used to detect but also to identify substances, molecules and the like based on their specific Raman spectrum. The Raman spectrum is achieved by spectrally resolving the inelastically scattered light from the sample. Thereafter, the measured spectrum is evaluated by matching it with the Raman spectra of known substances or microorganisms.
In comparison to fluorescence-based detection, Raman-spectroscopic detection has the advantage that no labelling of the sample is required and that the specificity of the Raman spectra associated to the various microorganisms is more pronounced than the specificity of the associated (auto-) fluorescence spectra.
As the Raman signal is usually orders of magnitude smaller than a fluorescence signal and as almost biological samples exhibit some degree of luminescence, such as for example fluorescence, the detection and separation of the Raman signal is comparably demanding. Also, maintaining a sufficiently good signal quality in Raman spectroscopic measurements becomes even more difficult when the amount of the sample is small.
In order to increase the signal-to-noise ratio of a Raman measurement, a first measure is to increase the illumination power of the illumination source, e.g. the laser. However, when measuring biological samples the amount of energy deposited on the sample during exposition to the laser light has to be limited in order not to affect or deteriorate the sample.
Another way to increase the signal-to-noise ratio for microscopic measurements is to perform these measurements using a so-called confocal detection scheme.
In such confocal microscopic Raman-spectroscopic measurements, the detection limit can be pushed to smaller amounts of sample. Also, the tighter the excitation light is focused on the sample, the stronger and more restrictive the confocality can be chosen and consequently the signal-to noise ratio is increased even more.
However, as these measurements with a tightly focused excitation light require high numerical aperture objective lenses, the working distances of these microscopes is strongly reduced, such that the objective lens and the sample have to be arranged in very close proximity. The working distance for example of a 100× oil immersion objective lens with a numerical aperture of 1.4 lies in the sub-millimeter range.
Furthermore, as the optics of such objective lenses are rather complex, refractive index changes in the light path will lead to aberrations of the focused laser light. Plastic covers of the sample chamber, air or other materials for example will cause such refractive index changes. Optical aberrations in turn always lead to an increased focal volume and thus to a deteriorated signal-to noise ratio.
However, in many potential applications, sterility conditions have to be met. Contamination of the sample by the environment or contamination of the environment by the sample is to be avoided. Thus, the use of a closed sample chamber or a sample chamber comprising a lid is mandatory. Furthermore, as biological samples are often grown in Petri dishes or contact plates that contain nutrition medium or a nutrition gel on the bottom, it is impossible to optically access the sample from the bottom of such a dish or plate, while maintaining a sufficiently good signal quality.
One solution to the problem is to house the whole confocal systems inside a closed atmosphere in order to avoid contamination of the sample.
Such housed systems however are comparably expensive and elaborate to handle.